A single amino acid substitution in lactate dehydrogenase improves the catalytic efficiency with an alternative coenzyme.
Using site-directed mutagenesis, the NADH-linked lactate dehydrogenase from Bacillus stearothermophilus has been specifically altered at a single residue to shift the coenzyme specificity towards NADPH. The single change is at position 53 in the amino acid sequence where a conserved aspartate has been replaced by a serine. This substitution was made to reduce steric hindrance on binding of the extra phosphate group of NADPH and to remove the negative charge of the aspartate group. The resultant mutant enzyme is 20 times more catalytically efficient than the wild-type enzyme with NADPH.
Feeney
R
R
Department of Biochemistry, University of Bristol, School of Medical Sciences, U.K.
Clarke
A R
AR
Holbrook
J J
JJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D004252:DNA Mutational Analysis; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D013329:Structure-Activity Relationship
D009243:NAD; D009249:NADP; D007770:L-Lactate Dehydrogenase